麦角硫因在游动放线菌底盘中的高效合成

Efficient synthesis of ergothioneine in Actinoplanes sp.

摘要:

【目的】 麦角硫因是一种稀有的天然氨基酸类强抗氧化剂，在机体内发挥着重要的生理功能，并在食品、医药、化妆品领域得到广泛应用。然而，传统的从蕈菇中提取和化学合成的方法存在收率低、成本高等问题，本研究期望利用代谢工程改造，提高游动放线菌(Actinoplanes sp.) HS中麦角硫因的产量。【方法】 首先通过生物信息学分析，锁定了Actinoplanes sp. HS中可能参与麦角硫因合成的基因；接着在大肠杆菌(Escherichia coli) BL21(DE3)中进行异源表达，进一步鉴定了这些基因的功能；最后将鉴定功能的基因组合后在Actinoplanes sp. HS中进行高表达，对突变株产量进行测定，并通过在发酵培养基中添加不同浓度的前体，探究其对发酵产量的影响。【结果】 Actinoplanes sp. HS中基因BC03-04016、BC03-04015、BC03-04014、BC03-04013所编码的酶可以合成麦角硫因前体组氨酸甜菜碱半胱氨酸亚砜(hercynine-cysteine-sulphoxide, HER-Cys-Sul)，基因BC03-04046和BC03-04917所编码的酶都具有裂解碳硫键的功能，可以催化前体HER-Cys-Sul形成终产物麦角硫因。将编码麦角硫因合成的基因高表达后得到的突变株YC313和YC314，其麦角硫因产量分别达到125 mg/L和108 mg/L，较出发菌株Actinoplanes sp. HS分别提高至2.9倍和2.5倍。在发酵培养基中额外添加0.35 g/L的甲硫氨酸后，YC313菌株的麦角硫因产量相较于原始培养基提高了24%；额外添加10 g/L的黄豆饼粉后，相较于原始培养基麦角硫因产量提高了19%。【结论】 本研究对Actinoplanes sp. HS中麦角硫因合成的相关基因及其功能进行了鉴定，通过代谢工程改造得到了两株麦角硫因高产菌株，并进一步研究了前体供应对麦角硫因产量的影响，为以游动放线菌为底盘生产麦角硫因提供了策略支持。

Abstract:

[Objective] Ergothioneine, a rare natural amino acid, is a powerful antioxidant with important physiological functions in the body. It has been widely used in the fields of food, medicine, and cosmetics. However, extracting ergothioneine from mushrooms and chemical synthesis suffer from low yields and high costs. This study aims to use metabolic engineering approaches to improve the yield of ergothioneine in Actinoplanes sp. HS. [Methods] Firstly, we locked onto the genes potentially involved in ergothioneine synthesis in Actinoplanes sp. HS by bioinformatics analysis. Then, we identified the functions of these genes by heterologous expression in Escherichia coli BL21(DE3). Finally, the identified functional genes were combined and overexpressed in Actinoplanes sp. HS, and the yields of ergothioneine in the mutant strains were measured. By adding different concentrations of precursors to the fermentation medium, we investigated the impact of precursor concentration on the yield of ergothioneine. [Results] The enzymes encoded by BC03-04016, BC03-04015, BC03-04014, and BC03-04013 in Actinoplanes sp. HS could synthesize the ergothioneine precursor hercynine-cysteine-sulphoxide (HER-Cys-Sul). The enzymes encoded by BC03-04046 and BC03-04917 had the function of cleaving carbon-sulfur bonds, which could catalyze the formation of the final product ergothioneine from HER-Cys-Sul. The mutant strains YC313 and YC314 obtained through overexpression of genes in ergothioneine synthesis showed ergothioneine yields of 125 mg/L and 108 mg/L, respectively, which were 2.9 times and 2.5 times that of the wild-type strain Actinoplanes sp. HS. Additional supplementation of 0.35 g/L methionine in the fermentation medium increased the ergothioneine yield of YC313 by 24% compared with that in the original medium, and the addition of 10 g/L soybean meal resulted in a 19% increase in the ergothioneine yield. [Conclusion] In this study, we identified the genes and their functions in ergothioneine synthesis in Actinoplanes sp. HS and obtained two strains with high yields of ergothioneine through metabolic engineering. Furthermore, we investigated the impact of precursor supply on the yield of ergothioneine, which provided strategic support for the production of ergothioneine by Actinoplanes sp.